Morphology, Biochemical, and Molecular Characterization of Pasteurella multocida Causing Hemorrhagic Septicemia in Indonesia

Pasteurella multocida is a Gram-negative bacterium that causes hemorrhagic septicemia (HS) in buffaloes and cattle. The disease causes serious problems in Indonesian livestock and is classified as a serious transmissible animal disease. Previous research has determined the diversity of P. multocida using a serotyping method based on the antigenic properties of capsule polysaccharides. An alternative method for analysis utilizes sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and random amplified polymorphic DNA (RAPD). This study aimed to characterize and determine P. multocida diversity in several regions of Indonesia based on phenotypic character, protein profile, and the band pattern of RAPD results. Bacterial identification was performed using traditional biochemical techniques and API® 20NE systems and then confirmed molecularly using polymerase chain reaction (PCR). The freeze-thawing technique was performed to obtain the bacterial protein extract, and DNA extraction was executed using DNAzol. The extracted protein and RAPD product were then electrophoresed on 12% polyacrylamide gel and 1.5% agarose gel, respectively. The results indicate that the molecular weight range of the protein bands is 12–209 kDa, and the band pattern of the RAPD results ranged from 307–3,100 bp. Based on phenotypical analysis, P. multocida from South Sulawesi Province exhibited a variety of growth characteristics in MacConkey agar media. Using the hierarchical clustering analysis of the band patterns of RAPD and the whole-cell protein profiles, four and five clusters were formed, respectively. These results indicate molecular diversity among P. multocida from several regions of Indonesia.


Introduction
Pasteurella multocida is an aerobic, Gram-negative bacteria with a short rod or coccoid structure. It is approximately 0.2-0.4 × 0.6-2.5 µm in size and is classifed as encapsulated (usually in virulent strains), nonmotile, and nonsporeforming. Furthermore, it exhibits a bipolar appearance in Gram staining. Te most signifcant biochemical characteristics for identifcation include indole production, nonhemolysis against sheep blood, positive oxidase reactions, lack of growth on MacConkey agar, and fermenting hexose sugar groups [1]. In ruminant species, P. multocida causes hemorrhagic septicemia (HS), whereas in avian species, it causes fowl cholera, and in swine, it causes atrophic rhinitis [2]. Between 2005 and 2019, there were 105,692,984 instances of HS reported across 41 nations. Te total number of deaths was 99,550, accounting for 0.1% of all cases and costing USD 692,092.90 yearly [3]. In Indonesia, HS is classifed as a serious transmissible animal disease by the Ministerial Decree of the Minister of Agriculture of the Republic of Indonesia, number 4026/KPTS/ OT.140/4/2013.
Te diversity of P. multocida is commonly detected through serotyping. Te serotyping classifcation is based on the antigenic properties of the capsule polysaccharide and the lipopolysaccharide (LPS) composition. A passive hemagglutination test has been used to classify P. multocida into fve serogroups (A, B, D, E, and F) based on the polysaccharide capsule structure, and a gel difusion precipitation test has classifed it into 16 serotypes (1-16) based on the composition of LPS [4][5][6][7][8]. However, this method has the disadvantage of being time-consuming and requiring specifc antisera. Tese specifc antisera are difcult to produce, especially the antisera against capsule polysaccharides, because they are less immunogenic [2,8]. Te alternatives for biodiversity analysis include the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and random amplifed polymorphic DNA (RAPD). Tis method has been demonstrated using various species of microorganisms. Previous studies have shown that SDS-PAGE can be used to conduct biodiversity analysis based on bacterial protein profles [9][10][11][12]. Terefore, the diversity of P. multocida could also be detected using RAPD [13][14][15][16].
It is necessary to analyze the diversity of P. multocida because research has long established that Indonesia possesses a rich biodiversity of microorganisms [17]. Terefore, the diversity of P. multocida from Indonesia is expected to be high. To the best of our knowledge, there has been no previous research on the diversity of P. multocida from Indonesia based on RAPD patterns and whole-cell protein profles. Tis characterization is important because it can serve as a cornerstone for studying the diversity of P. multocida from Indonesia and predicting its virulence profle. Furthermore, these fndings can be utilized in developing an immunodiagnostic kit because protein, one of the antigenic components of P. multocida [2,[18][19][20][21], is commonly used in the development of immunodiagnostic kits.

Cultural and Biochemical Identifcation of P. multocida.
Ten P. multocida isolates from the Indonesian Research Center for Veterinary Science's collection were used in this study, consisting of nine isolates from several regions in Indonesia and one standard isolate of P. multocida B:2 (Table 1). Tese isolates were cultured on 5% sheep blood agar (SBA) (Oxoid Ltd., UK) and MacConkey agar (Oxoid Ltd., UK), followed by incubation at 37°C for 24 h. Te plates were then examined for growth. Next, the colonies were Gram stained and tested, then observed for the following characteristics: no growth on MacConkey agar, catalase and oxidase positivity, indole production, nonmotility, nonhemolysis on SBA, and acid from glucose [22,23]. Biochemical identifcation was also performed using an API ® 20NE kit (bioMérieux, France) based on its instruction manual.

DNA Extraction.
Genomic DNA was extracted using DNAzol (Molecular Research Center, Inc., USA) and preserved at −20°C until further analysis.

Identifcation of P. multocida Based on 23S rRNA and ompH
Genes. Te 23S rRNA and ompH genes were amplifed in PCR using oligonucleotide primers, using the methods previously described by Mifin and Blackall [24] and Luo et al. [25], respectively ( Table 2). PCR was performed using 20 ng of genomic DNA along with forward and reverse primers (10 pmol each) in a MyTaq HS Red Mix (Bioline, USA) master mix. Te PCR-amplifed product was analyzed on a 1% agarose gel along with a DNA molecular weight marker.

RAPD.
Te RAPD amplifcation was performed using M13 primer based on Taylor et al.'s method [26] (Table 2), using 20 ng of genomic DNA and 10 pmol of primer in an i-Taq master mix solution (Intron, Republic of Korea). Te RAPD product was analyzed on a 1.5% agarose gel along with a DNA molecular weight marker.

Protein Extraction.
Bacterial whole-cell protein was extracted based on the modifed method described by Rachmawati et al. [27]. In brief, bacterial cells were washed twice with phosphate-bufered saline by centrifugation at 7,500 × g for 15 minutes (M-Science, MPW Med. Instruments, Poland). Te soluble protein was obtained by freeze-thawing the bacterial suspension in liquid nitrogen, and the suspension was centrifuged at 920 × g for 10 minutes. Te supernatant was then collected and preserved at −20°C until further analysis.

Protein Electrophoresis (SDS-PAGE).
Electrophoresis was performed on a 12% polyacrylamide gel (Invitrogen, USA) using the method by Laemmli [28]. In short, 10 µg protein and a Laemmli sample bufer (Bio-Rad, USA) were homogenized at a 1 : 1 ratio. Protein samples, along with a protein molecular weight marker (Termo Scientifc, Lithuania), were electrophoresed at a constant voltage of

Bacterial Identifcation.
All isolates exhibited growth on SBA, and nonhemolysis was observed (Figure 1(a)). Te bacterial cells' characteristics were Gram-negative with a coccoid bipolar structure (Figure 1(b)). All isolates were also observed to be nonmotile, catalase-and oxidasepositive, and indole positive, and they were all capable of producing acid from glucose. Eight isolates exhibited no growth on MacConkey agar, and they were correctly identifed as P. multocida by API ® 20NE with a numerical profle of 3000004 (% ID � 96%). Tis numerical profle is consistent with the results of previous studies [29,30]. Meanwhile, the other two isolates (468 and 492) grew pink bacterial colonies on MacConkey agar (Figure 1(c)). Interestingly, the results of API ® 20NE identifed these two isolates as Vibrio parahaemolyticus, with a numerical profle of 7067746 (% ID � 98.9%), and Aeromonas hydrophila, with a numerical profle of 7467746 (% ID � 87%), respectively. However, molecular identifcation by PCR demonstrated that all the isolates used were P. multocida (Figures 2(a) and 2(b)).

P. multocida Diversity Based on RAPD.
RAPD showed variations in the pattern of DNA fragments against the 10 isolates of P. multocida used in this study. Tere were 6-8 bands generated in these fragment patterns, with a range ranging from 307-3,100 bp (Figure 3(a)). Hierarchical clustering analysis of DNA fragment patterns indicated the formation of four clusters, using a 95% similarity value (Figure 3(b)). Tese results are in line with the results of

P. multocida Diversity Based on Protein
Profle. SDS-PAGE results indicated that the whole-cell protein band range of P. multocida isolates used in this study ranged from 12-209 kDa (Figure 4(a)). Hierarchical clustering analysis of protein profles grouped 10 isolates into fve clusters with a similarity value of 70% (Figure 4(b)).
Tese results are consistent with previous studies: protein profle dendrograms with similarity values above 65% in P. multocida [32] and 70% in coagulase-negative staphylococci are considered sufciently high for observing intraspecies diversity [11]. In this clustering, the isolates that were able to grow on MacConkey agar were grouped into one cluster (cluster III), and the other eight isolates were divided into four additional clusters. Four isolates from Lampung Province, namely, B2951, B2953, B2954, and B2955, were separated into two diferent clusters. In general, clustering based on protein profles by SDS-PAGE and DNA fngerprinting by RAPD shows similar results.

Discussion
P. multocida exhibits a lack of growth on MacConkey agar. However, on rare occasions, it is still possible for several strains to grow on MacConkey agar. Tis evidence was reported by Heddleston and Wessman in a previous study [33], which found that one P. multocida isolate could grow on MacConkey agar out of a total of 1,088 isolates tested.  Figure 2: Results of PCR amplifcation targeting the 23S rRNA (a) and ompH (b) genes in P. multocida. Te 23S rRNA gene is one of the target genes that can be used to identify P. multocida. Its amplifcation product size is approximately 1,400 bp [24]. Te ompH gene is a virulent gene that belongs to P. multocida, with an amplifcation product size of approximately 1,000 bp [25]

Veterinary Medicine International
Pink colonies also indicate that these bacteria exhibit the property of being able to ferment lactose. Tis characteristic is rarely found in P. multocida, but it can occasionally occur [34], as confrmed by the results of previous studies [33][34][35][36][37]. Cases of lactose fermentation in P. multocida generally occur in P. multocida biovars 12 and 14 [36].
Cases of misidentifcation in API ® 20NE are most likely caused by the indicators used in its identifcation system. Such indicators are mostly derived from information concerning the ability of bacteria to utilize carbohydrates in their biological systems. If two bacterial species have similar carbohydrate utilization characteristics, API ® 20NE will most likely misidentify the two species as the same species. Tis error will be problematic if this kit is used exclusively for identifcation, especially if the results provide a high identifcation percentage value, as in the results of this study.
As in previous studies, the identifcation results of the API ® kit were deemed accurate in this study if the identifcation percentage value exceeded 80% [38].
Te biochemical characteristics of Aeromonas bacteria-especially those related to carbohydrate utilization-are similar to P. multocida [39]. Te characteristics that can be used to distinguish Pasteurella from Aeromonas and Vibrio are their motility and type of hemolysis. Aeromonas hydrophila and Vibrio parahaemolyticus are motile bacteria [40,41]. Aeromonas bacteria generally have the β-hemolysis type, while P. multocida have the nonhemolysis type [39]. Both of these characteristics are not accommodated by the API ® 20NE test, so they can cause misidentifcation if not tested separately. Cases of misidentifcation by API ® 20NE on P. multocida have also been reported in a previous study [30]. Te misidentifed isolate was identifed as Aeromonas salmonicida (% ID � 55.2%). Te study also found P. multocida isolates that were misidentifed as Vibrio hollisae (% ID � 80.6%) using API ® 20E. Other studies indicate that the API ® identifcation system is not infallible, but its efectiveness still exceeds 80% [42][43][44]. Cumulatively, these fndings support previous reports that the API ® 20NE kit yields inconsistent identifcation percentages for the same organism. As such, the API ® 20NE kit should be used to complement traditional identifcation systems, thereby minimizing the risk of misidentifcation.
Te RAPD clustering performed in this study also supports the results of previous studies [26,31,45]. Te present study's method demonstrates that the M13 primer can be used for genetic diferentiation (DNA fngerprinting) of P. multocida and can thus describe the diversity among P. multocida isolates from Indonesia. Te dendrogram also indicates the diversity among P. multocida isolates from the same region. Isolates from Lampung Province and South Sulawesi Province were grouped into two diferent clusters (clusters II and III for Lampung Province; clusters II and IV for South Sulawesi Province). Previous research in Brazil demonstrated that 14 P. multocida isolates sourced from the city of Diamantino could be grouped into two diferent clusters when analyzed using primer M13 [31]. Findings in other countries also indicate that P. multocida originating from one region exhibits high diversity when analyzed using RAPD. In India, P. multocida isolated from the Tamil Nadu region exhibited diversity that could be grouped into nine clusters [46]. Similar fndings were also reported in the Republic of Korea, where P. multocida isolated from Gyeongsangbuk-do Province exhibited diversity that could be grouped into eight clusters [45]. Tese results indicate that RAPD is a simple and fast method for identifying patterns that are useful for strain diferentiation [46].
Te dendrogram based on SDS-PAGE also demonstrates the diversity of P. multocida from several regions in Indonesia. In cluster IV, isolates B2953 and B2954 were joined in one cluster with M1404. In this cluster, B2954 and M1404 exhibited the highest similarity level (94.68%). Meanwhile, isolates B2951 and B2955 were grouped in one cluster with isolate 471 (cluster II), which originated from South Sulawesi Province; these had a similarity level of    [32], the clusters can also be assumed to refect the isolates' regions of origin. Furthermore, the diversity of protein profles has consequences for the diversity of immunogenic proteins. Tese proteins can trigger antibody responses and be detected by a Western blot test [47]. Te identifcation of isolates using the phenotypic, biochemical, and physiological characteristics of microorganisms is frequently impractical and inefcient, and the results are sometimes unreliable due to variations in the reactions from one test to the next [10,11]. Te classifcation method used for P. multocida serotypes is usually performed using time-consuming serological methods [4,5], and the specifc antisera are difcult to produce. SDS-PAGE is advantageous because it can be completed in one day, unlike conventional phenotypic methods. Information about the estimated genome of a microorganism can also be derived simply and quickly using a whole-cell protein profle. Whole-cell protein profles also correlate with the biochemical characteristics of a species because diferent physiological characteristics tend to produce diferent protein profles [10]. Te results of this study support this assertion by illuminating the striking diferences in RAPD and protein profles between isolates that can grow on MacConkey agar and those that cannot.
Te diversity of isolates from the same region is also observable based on their protein profles. Isolates from Lampung Province and South Sulawesi Province were grouped into two clusters. Similar to the RAPD results, this fnding indicates that P. multocida isolates from the same region are likely to be diverse. Similar fndings have also been reported in a previous study [48], in which several P. multocida isolates from the same region had diferent protein profles. For another example, isolates of Trypanosoma evansi from the same village in South Kalimantan Province had diverse protein profles [49].
Tis study's fndings can serve as an initial guideline for the development of an immunodiagnostic kit. As previously reported [47], the selection of isolates based on protein profles and the characteristics of immunogen proteins as antigen candidates constitute important steps in the development of an enzyme-linked immunosorbent assay. Subekti and Yuniarto [47] reported that isolates with different protein profles may yield a diferent image of specifc immunogenic proteins when tested against the same standard sera. Tese isolates may even exhibit diferent nonspecifc reactions or cross-reactions to the same negative standard serum. Te same phenomenon has also been reported in other studies [21,50]. Tese fndings indicate that not all isolates are suitable for use as antigens in the development of an immunodiagnostic kit. Te existence of diversity among P. multocida isolates from various regions of Indonesia is important initial information to guide the development of an immunodiagnostic kit and allow for further exploration of these isolates. Genome sequencing is recommended for analysis of sequence-based similarities. In addition, analysis of immunogenic proteins using a Western blot test also needs to be carried out at a later stage in the development of an immunodiagnostic kit.

Conclusion
In conclusion, the molecular weight range of the protein bands of P. multocida from several regions in Indonesia is 12-209 kDa, and the band pattern of the RAPD results ranged from 307-3,100 bp. Using the hierarchical clustering analysis of the band patterns of RAPD and the whole-cell protein profles, four and fve clusters were formed, respectively. Tese results indicate molecular diversity among P. multocida from several regions of Indonesia.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.